PROJECT SUMMARY Protein misfolding and disruption of normal protein homeostasis play critical roles in many neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). Protein dyshomeostasis is an important contributor to aging, the most important risk factor for neurodegenerative disease. Progranulin (GRN) is a conserved protein with links to normal aging and neurodegeneration. Haploinsufficiency causes FTD, nullizygosity causes neuronal ceroid lipofuscinosis?a lysosomal storage disorder, and polymorphisms in the GRN gene may increase the risk of common neurodegenerative diseases, including AD. We hypothesize that GRN deficiency leads to neurodegeneration by impairing the autophagy/lysosomal pathway (ALP), a critical arm of the proteostasis network (PN), and that ALP deficits contribute to neurodegenerative diseases more broadly. We performed a genome-wide screen to discover modifiers of GRN levels and hits enriched in the ALP. We found a reciprocal relationship between GRN and the ALP in primary neurons: GRN levels were regulated by autophagy and GRN deficiency impaired lysosomes and inhibited ALP flux. Interestingly, genetic modifiers from our screen that raised GRN levels rescued lysosomal and autophagy deficits caused by GRN deficiency. Our project will investigate the mechanistic relationship between ALP function and neurodegeneration and explore ALP as a therapeutic target. In Aim 1, we will study how GRN deficiency affects the ALP and other PN components. We will use proteomics, lipidomics, and RNAseq to define how GRN deficiency alters cell and lyososome physiology. In addition, we will use an innovative high-throughput longitudinal single-cell analysis platform, robotic microscopy, with biosensors for ALP and other arms of PN to determine how GRN deficiency affects their function dynamically and in response to stress. In Aim 2, we will investigate how GRN deficiency and ALP dysfunction lead to accumulation of Tar DNA binding protein 43 (TDP43) and affect the metabolism of other proteins linked to neurodegenerative disease. In Aim 3, the Kelly and Finkbeiner labs will collaborate to identify new small molecules to modulate the autophagy pathway. In preliminary studies, novel autophagy inducers were identified that promote the clearance of several disease-causing proteins (e.g., tau, synuclein, TDP43 and mutant huntingtin) and mitigate neurodegeneration phenotypes in human neurons differentiated from iPSCs of patients with ALS and HD. The discovery of new, safe and effective small-molecule autophagy inducers would be useful research tools and may form the basis for new therapeutic approaches to neurodegenerative disease. This project will synergize with the broader program by providing expertise and novel assays of the ALP, innovative iPSC models of neurodegenerative disease and new tools to modulate the ALP. Collaboration with other projects in the program will help this project put ALP in the context of the broader proteostasis network.